RNA and DNA purification...2 Lyse cells 350 μL RA1, RAP, or RP1 3.5 μL reducing agent Mix 100 μL RA1 2 μL TCEP Mix 5 μL Carrier RNA 3 Filtrate ysatel 11,000 x g 1 min 11,000 x

US:Tel.: +1 484 821 0984 Fax: +1 484 821 1272 E-mail: [email protected]

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FR:Tel.: +33 388 68 22 68 Fax: +33 388 51 76 88 E-mail: [email protected]

MACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · Deutschland

www.mn-net.com

www.mn-net.comDE / International:Tel.: +49 24 21 969-0Fax: +49 24 21 969-199 E-Mail: [email protected]

CH:Tel.: +41 62 388 55 00 Fax: +41 62 388 55 05 E-Mail: [email protected]

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RNA and DNA purification

User manualNucleoSpin® RNA / DNA Buffer Set

January 2018 / Rev. 10

frieds

MN Stamp 2018

MACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · GermanyTel.: +49 24 21 969-270 · Fax: +49 24 21 969-199 · [email protected] · www.mn-net.com

RNA and DNA purificationProtocol-at-a-glance (Rev. 10)

NucleoSpin® RNA / miRNA / RNA Blood / RNA Plant, NucleoSpin® RNA/Protein NucleoSpin® RNA XS

1 Homogenize sample

Sample Sample

2 Lyse cells 350 μL RA1, RAP, or RP1 3.5 μL reducing agent

Mix

100 μL RA1 2 μL TCEP

Mix

5 μL Carrier RNA

3 Filtrate lysate 11,000 x g

1 min

11,000 x g 30 s

4 Adjust RNA binding conditions

350 μL 70 % ethanol

Mix

100 μL 70 % ethanol

Mix

5 Bind RNA / DNA Load lysate

11,000 x g 30 s

Load lysate

11,000 x g 30 s

A Wash silica membrane

1st wash

2nd wash

500 μL DNA Wash

500 μL DNA Wash

11,000 x g 1 min

400 μL DNA Wash

400 μL DNA Wash

11,000 x g 1 min

Nuc

leoS

pin®

RN

A/D

NA

Buf

fer

Set

B Dry membrane RT, 3 min RT, 3 min

C Elute DNA 100 μL DNA Elute

11,000 x g 1 min

80 μL DNA Elute

11,000 x g 1 min

7 Digest DNA 95 μL DNase reaction mixture

RT, 15 min

25 μL DNase reaction mixture

RT, 15 min

8 Wash and dry silica membrane

1st and 2nd

3rd

1st wash

2nd wash

3rd wash

200 μL RA2

600 μL RA3

250 μL RA3

11,000 x g 30 s

11,000 x g 2 min

100 μL RA2

400 μL RA3

200 μL RA3

11,000 x g 30 s

11,000 x g 2 min

9 Elute highly pure RNA

60 μL RNase-free H2O

11,000 x g 1 min

10 μL RNase-free H2O

11,000 x g 30 s

3MACHEREY-NAGEL – 01/2018, Rev. 10


Table of contents1 Components 4

1.1 Set contents 4

1.2 Consumables and equipment to be supplied by user 4

1.3 About this user manual 4

2 Product description 5

2.1 The basic principle 5

2.2 Kit specifications 6

3 Storage conditions and preparation of working solutions 8

4 Safety instructions 8

5 Protocol – isolation of RNA and DNA from one undivided sample 9

6 Appendix 11

6.1 Troubleshooting 11

6.2 Ordering information 12

6.3 Product use restriction / warranty 13

MACHEREY-NAGEL – 01/2018, Rev. 104


1 Components

1.1 Set contents

NucleoSpin® RNA/DNA Buffer Set

REF100 preps

740944

Buffer DNA Wash (Concentrate)* 2 x 12 mL

Buffer DNA Elute 12 mL

User manual 1

1.2 Consumables and equipment to be supplied by userThe content of this set is sufficient for 100 DNA isolations in combination with RNA isolations performed with the following kits:

NucleoSpin® RNA (REF 740955), NucleoSpin® miRNA (REF 740971), NucleoSpin® RNA Blood (REF 740200), NucleoSpin® RNA Plant (REF 740949), NucleoSpin® RNA/Protein (REF 740933), NucleoSpin® RNA XS (REF 740902), NucleoSpin® 8 RNA (REF 740698), NucleoSpin® 8 RNA Core Kit (REF 740456), NucleoSpin® 96 RNA (REF 740709), NucleoSpin® 96 RNA Core Kit (REF 740466).

Additional collection tubes are required and are not supplied (see section 6.2, ordering information).

1.3 About this user manualIt is strongly recommended reading the detailed protocol sections of this user manual if the NucleoSpin® RNA/DNA Buffer Set is used in combination with NucleoSpin® RNA (REF 740955), NucleoSpin® miRNA (REF 740971), NucleoSpin® RNA Blood (REF 740200), NucleoSpin® RNA Plant (REF 740949), NucleoSpin® RNA/Protein (REF 740933), NucleoSpin® RNA XS (REF 740902) NucleoSpin® 8 RNA (REF 740698), NucleoSpin® 8 RNA Core Kit (REF 740456), NucleoSpin® 96 RNA (REF 740709), or NucleoSpin® 96 RNA Core Kit (REF740466) for the first time. Experienced users, however, may refer to the Protocol at a glance instead. The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure.

All technical literature is available on the internet at www.mn-net.com.

* For preparation of working solutions and storage conditions see section 3.



2 Product description

2.1 The basic principleThe NucleoSpin® RNA/DNA Buffer Set is intended to be used with one of the following RNA purification kits: NucleoSpin® RNA, NucleoSpin® miRNA, NucleoSpin® RNA Blood, NucleoSpin® RNA Plant, NucleoSpin® RNA / Protein, NucleoSpin® RNA XS NucleoSpin® 8 RNA, NucleoSpin® 8 RNA Core Kit, NucleoSpin® 96 RNA , or NucleoSpin® 96 RNA Core Kit. The combination the NucleoSpin® RNA/DNA Buffer Set with either of the RNA purification kits enables the isolation of RNA and DNA from one undivided sample with one single NucleoSpin® RNA Binding Column. This patented technology enables successive elution of DNA and RNA from a NucleoSpin® Column with low salt buffer and water respectively. DNA and RNA are immediately ready for downstream applications. Samples are lysed in the lysis buffer supplied in the NucleoSpin® RNA kits (Lysis Buffer RA1, RAP, or RP1). Ethanol is added to facilitate conditions for binding of nucleic acids to the NucleoSpin® RNA Binding Column. After wash steps DNA and RNA are eluted sequentially. DNA is eluted with a low salt solution (DNA Elute) which selectively elutes DNA and keeps RNA on the column. Eluted DNA is immediately ready for downstream applications without further purification. DNA eluted with DNA Elute may readily serve as template for PCR, is restrictable with restrictions enzymes and is of high molecular weight (≥ 20 kb). A260 / A280 ratios of eluted DNA are within a range from 1.7–2.0.

After DNA elution, residual on-column-DNA is digested on the NucleoSpin® Column as described in the relating NucleoSpin® RNA protocol. After additional washing steps, pure RNA is eluted with RNase-free water. DNA elution prior to RNA elution does neither compromise RNA quality nor quantity. Sequential DNA and RNA isolation from one sample with this support set and NucleoSpin® RNA kits has been successfully performed with various sample materials (e.g., HeLa cells, pig liver, kidney and spleen, parsley leaf, maize leaf, and root).

The standard protocol (section 5) allows the purification of DNA and RNA from a variety of sample types. Suitable sample types are described in the respective user manuals of the NucleoSpin® RNA kits.



2.2 Kit specificationsTypical yields of RNA and DNA

0.1

1

10

100

10,000 100,000 1,000,000

HeLa cell number

DNA, RNA yield [µg]RNA

DNA D

NA

, RN

A y

ield

[μg]

Figure 1 DNA and RNA yield from different amounts of HeLa cells Different amounts of HeLa cells were used as sample material. DNA and RNA were isolated with the NucleoSpin® RNA/DNA Buffer Set in combination with the NucleoSpin® RNA kit.

DNA and RNA were isolated as described in Figure 1. Obtained correlation coefficients between sample amount and RNA and DNA yield are shown in Table 1.

Table 1: Correlation between sample amount and nucleic acid yield

3 x 104–5 x 105 cells 3 x 104–1 x 106 cells

RNA > 0.98 > 0.98

DNA > 0.99 > 0.95



0.01

0.1

1

10

100

0.01 0.1 1 10

Mouse liver [mg]

DN.NA yield [µg]

RNA

DNA D

NA

, RN

A y

ield

[μg]

Figure 2 DNA and RNA yield from different amounts of mouse liver tissue Different amounts of mouse liver tissue were used as sample material. DNA and RNA were isolated with the NucleoSpin® RNA/DNA Buffer Set in combination with the NucleoSpin® RNA kit.

DNA and RNA were isolated as described in Figure 2. Obtained correlation coefficients between sample amount and RNA and DNA yield are shown in Table 2.

Table 2: Correlation between sample amount and nucleic acid yield

0.08–1.25 mg mouse liver

0.08–2.5 mg mouse liver

0.08–5 mg mouse liver

RNA > 0.98 > 0.98 > 0.98

DNA > 0.99 > 0.95 > 0.67

DNA size and quality

• Isolated genomic DNA is commonly of high molecular weight > 20 kb.

• DNA is commonly stable, even at 37 °C for 2 h with or without addition of a typical restriction enzyme buffer.

• DNA is digestable with restriction enzymes.

• DNA is suitable for PCR.



3 Storage conditions and preparation of working solutions

Store solutions at room temperature (18–25 °C).

• The DNA Wash solution is delivered as a concentrate. To prepare the final DNA Wash solution, add four volumes of ethanol (50 %) to the DNA Wash Concentrate (add 48 mL 50 % ethanol to 12 mL DNA Wash Concentrate).

• Due to its composition DNA Elute (DNA elution buffer) does not inhibit DNases, i.e., DNA Elute does not contain substances (e.g., EDTA) to complex divalent cations. Therefore, make sure not to contaminate DNA Elute with DNases!

• Further, due to its composition, DNA Elute does not inhibit microbial growth. Therefore, make sure not to contaminate DNA Elute with any source of microbial contaminants.


REF100 preps

740944

Buffer DNA Wash (Concentrate) 2 x 12 mL Add 48 mL ethanol (50 %) to each bottle

4 Safety instructionsThe NucleoSpin® RNA/DNA Buffer Set is intended to be used in conjunction with NucleoSpin® RNA kits. The NucleoSpin® RNA/DNA Buffer Set does not contain hazardous contents. However, pay attention to the safety instructions of the individual NucleoSpin® RNA kits!



5 Protocol – isolation of RNA and DNA from one undivided sample

Before starting the procedure:

• Check if Buffer DNA Wash was prepared according to section 3.

• Perform sample homogenization, cell lysis, lysate filtration, adjusting of nucleic acid binding conditions, and binding of nucleic acids to the NucleoSpin® RNA Binding Column according to the NucleoSpin® RNA, NucleoSpin® miRNA, NucleoSpin® RNA Blood, NucleoSpin® RNA Plant, NucleoSpin® RNA/Protein, NucleoSpin® RNA XS, NucleoSpin® 8 RNA, NucleoSpin® 8 RNA Core Kit, NucleoSpin® 96 RNA or NucleoSpin® 96 RNA Core Kit kit standard protocol.

Subsequent to binding of nucleic acids to the column continue as follows with step A (the membrane desalting step of the individual NucleoSpin® RNA protocols is replaced by steps A–C):

A Wash silica membrane

1st wash

Add 500 µL DNA Wash to the NucleoSpin® RNA Binding Column and centrifuge for 1 min at 11,000 x g. Discard flowthrough and reuse Collection Tube.

If using NucleoSpin® RNA XS add only 400 µL DNA Wash.

The DNA Wash solution is used instead of MDB (Membrane Desalting Buffer) from the NucleoSpin® RNA kits. MDB will not be used in this procedure.

2nd wash

Add again 500 µL DNA Wash and centrifuge 1 min at 11,000 x g. Discard Collection Tube with flowthrough.

If using NucleoSpin® RNA XS add only 400 µL DNA Wash.

+ 500 µL DNA Wash

11,000 x g, 1 min

+ 500 µL DNA Wash

11,000 x g, 1 min

B Dry membrane

Insert the NucleoSpin® RNA Binding Column into a new 1.5 mL microcentrifuge tube (not supplied). Open the lid of the NucleoSpin® RNA Binding Column and let it stand for 3 minutes.

The procedure ensures complete removal of ethanol from the column.

Incubate for 3 min



C Elute DNA

Add 100 µL DNA Elute (DNA elution buffer) directly onto the membrane and incubate 1 min. Elute the DNA by centrifuging for 1 min at 11,000 x g.

If using NucleoSpin® RNA XS add only 80 µL DNA Elute for elution.

Add 100 μL

DNA Elute

The temperature of the DNA Elute solution shall not exceed 30 °C, otherwise RNA will partly elute with the DNA Elute solution. DNA Elute solution may stay for 1 min up to 15 min on the column before DNA is eluted. A 1–5 min incubation time is recommended. Eluted DNA is immediately ready for downstream applications without further purification.

11,000 x g,

1 min

Proceed with the digestion of residual on-column DNA according to the individual NucleoSpin® RNA protocols (step: Digest DNA): Add DNase reaction mixture onto the column and perform all subsequent steps as described in the NucleoSpin® RNA, NucleoSpin® miRNA, NucleoSpin® RNA Blood, NucleoSpin® RNA Plant, NucleoSpin® RNA/Protein, NucleoSpin® RNA XS, NucleoSpin® 8 RNA, NucleoSpin® 8 RNA Core Kit, NucleoSpin® 96 RNA, or NucleoSpin® 96 RNA Core Kit protocol.



6 Appendix

6.1 Troubleshooting

Problem Possible cause and suggestions

DNA is contaminated with RNA

Buffer temperature

• DNA elution buffer DNA Elute exceeded 30 °C during application. Use DNA Elute with a temperature preferentially of 18–25 °C.

DNA yield lower than RNA yield

Sample material

• DNA and RNA yield depend very much on sample material. Ratio of RNA yield to DNA yield may vary from approximately 1–20.

DNA degrades upon storage

DNase contamination

• DNA elution buffer DNA Elute does not contain divalent cations complexing substances (e.g., EDTA). Therefore, DNA is not protected against DNases. Keep DNA Elute solution clean and avoid any contamination. As a precaution, keep DNA on ice for short term or at - 20 °C for long term storage

• Some sample materials may contain remaining DNase traces that are not sufficiently washed away by the standard procedure. Perform a wash step of the column with Buffer RA2 after loading the lysate onto the column and before starting the washing steps with DNA Wash solution: Add 500 µL Buffer RA2 onto the column, centrifuge 1 min at 11,000 x g and continue with DNA Wash washing steps.

Low RNA yield or quality

See general protocol

• See troubleshooting section of individual NucleoSpin® protocols. Check if Wash Buffer RA3 has been equilibrated to room temperature before use. Washing at lower temperatures lowers efficiency of salt removal by Wash Buffer RA3.

Suboptimal performance of DNA in downstream applications

Divalent cations

• Eluted DNA contains small amounts of divalent cations. If the downstream application comprises for example 50 % DNA eluate of the final reaction volume the divalent cations introduced into the reaction by the DNA eluate may alter the performance. Decrease the divalent cation concentration of the reaction by 1–5 mM for compensation.

Low DNA yield for large sample amounts

Sample amount too large

• Depending on the type of sample and its DNA content, DNA yield may not increase proportional with increased sample amount. Sample amounts larger than for example 5 mg tissue or 106 cultured cells may yield less DNA than smaller sample amounts. Use smaller sample to ensure good correlation between sample amount and DNA yield.



6.2 Ordering information

Product REF Pack of

NucleoSpin® RNA/DNA Buffer Set* 740944 100 preps

NucleoSpin® RNA 740955.10 / .50 / .250 20 / 50 / 250 preps

NucleoSpin® miRNA 740971.10 / .50 / .250 10 / 50 / 250 preps

NucleoSpin® RNA Blood 740200.10 / .50 10 / 50 preps

NucleoSpin® RNA Plant 740949.10 / .50 / .250 10 / 50 / 250 preps

NucleoSpin® RNA/Protein 740933.10 / .50 / .250 10 / 50 / 250 preps

NucleoSpin® RNA XS 740902.10 / .50 / .250 10 / 50 / 250 preps

NucleoSpin® TriPrep* 740666.10 / .50 / .250 10 / 50 / 250 preps

NucleoSpin® 8 RNA 740698 / .5 12 x 8 / 60 x 8 preps

NucleoSpin® 8 RNA Core Kit 40456.4 48 x 8 preps

NucleoSpin® 96 RNA 740709.2 / .4 / .24 2 x 96 / 4 x 96 / 24 x 96 preps

NucleoSpin® 8 RNA Core Kit 740466.4 4 x 96 preps

Buffer RA1 740961 50 mL

Buffer RA1 740961.500 500 mL

Buffer RP1 740934.50 50 mL

Buffer RP1 740934.500 500 mL

rDNase Set 740963 1 set

NucleoSpin® Filters 740606 50

NucleoSpin® 96 RNA Filter Plate 740711 4 plates

Collection Tubes (2 mL) 740600 1000

Visit www.mn-net.com for more detailed product information.

* DISTRIBUTION AND USE OF NUCLEOSPIN® RNA/DNA BUFFER SET and NUCLEOSPIN® TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS.



6.3 Product use restriction / warrantyNucleoSpin® RNA/DNA Buffer Set kit components are intended, developed, designed, and sold FOR RESEARCH PURPOSES ONLY, except, however, any other function of the product being expressly described in original MACHEREY-NAGEL product leaflets.

MACHEREY-NAGEL products are intended for GENERAL LABORATORY USE ONLY! MACHEREY-NAGEL products are suited for QUALIFIED PERSONNEL ONLY! MACHEREY-NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING. For detailed information please refer to the respective Material Safety Data Sheet of the product! MACHEREY-NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT. MACHEREY-NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application. Application on the human body is STRICTLY FORBIDDEN. The respective user is liable for any and all damages resulting from such application.

DNA/RNA/PROTEIN purification products of MACHEREY-NAGEL are suitable for IN VITRO-USES ONLY!

ONLY MACHEREY-NAGEL products specially labeled as IVD are also suitable for IN VITRO-diagnostic use. Please pay attention to the package of the product. IN VITRO-diagnostic products are expressly marked as IVD on the packaging.

IF THERE IS NO IVD SIGN, THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO-DIAGNOSTIC USE!

ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE (INCLUDING, BUT NOT LIMITED TO DIAGNOSTIC, THERAPEUTIC AND/OR PROGNOSTIC USE).

No claim or representations is intended for its use to identify any specific organism or for clinical use (included, but not limited to diagnostic, prognostic, therapeutic, or blood banking). It is rather in the responsibility of the user or - in any case of resale of the products - in the responsibility of the reseller to inspect and assure the use of the DNA/RNA/protein purification products of MACHEREY-NAGEL for a well-defined and specific application.

MACHEREY-NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in-house quality control, product documentation and marketing material.

This MACHEREY-NAGEL product is shipped with documentation stating specifications and other technical information. MACHEREY-NAGEL warrants to meet the stated specifications. MACHEREY-NAGEL´s sole obligation and the customer´s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted. Supplementary reference is made to the general business terms and conditions of MACHEREY-NAGEL, which are printed on the price list. Please contact us if you wish to get an extra copy.

There is no warranty for and MACHEREY-NAGEL is not liable for damages or defects arising in shipping and handling (transport insurance for customers excluded), or out of accident or improper or abnormal use of this product; defects in products or components not manufactured by MACHEREY-NAGEL, or damages resulting from such non-MACHEREY-NAGEL components or products.

MACHEREY-NAGEL makes no other warranty of any kind whatsoever, and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE



WHATSOEVER, DIRECTLY OR INDIRECTLY, EXPRESS OR IMPLIED, INCLUDING, WITHOUT LIMITATION, AS TO THE SUITABILITY, REPRODUCTIVITY, DURABILITY, FITNESS FOR A PARTICULAR PURPOSE OR USE, MERCHANTABILITY, CONDITION, OR ANY OTHER MATTER WITH RESPECT TO MACHEREY-NAGEL PRODUCTS.

In no event shall MACHEREY-NAGEL be liable for claims for any other damages, whether direct, indirect, incidental, compensatory, foreseeable, consequential, or special (including but not limited to loss of use, revenue or profit), whether based upon warranty, contract, tort (including negligence) or strict liability arising in connection with the sale or the failure of MACHEREY-NAGEL products to perform in accordance with the stated specifications. This warranty is exclusive and MACHEREY-NAGEL makes no other warranty expressed or implied.

The warranty provided herein and the data, specifications and descriptions of this MACHEREY-NAGEL product appearing in MACHEREY-NAGEL published catalogues and product literature are MACHEREY-NAGEL´s sole representations concerning the product and warranty. No other statements or representations, written or oral, by MACHEREY-NAGEL´s employees, agent or representatives, except written statements signed by a duly authorized officer of MACHEREY-NAGEL are authorized; they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty.

Product claims are subject to change. Therefore please contact our Technical Service Team for the most up-to-date information on MACHEREY-NAGEL products. You may also contact your local distributor for general scientific information. Applications mentioned in MACHEREY-NAGEL literature are provided for informational purposes only. MACHEREY-NAGEL does not warrant that all applications have been tested in MACHEREY-NAGEL laboratories using MACHEREY-NAGEL products. MACHEREY-NAGEL does not warrant the correctness of any of those applications.

Please contact: MACHEREY-NAGEL GmbH & Co. KG Tel.: +49 24 21 969-270 [email protected]

US:Tel.: +1 484 821 0984 Fax: +1 484 821 1272 E-mail: [email protected]

MACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · Germany

www.mn-net.com

www.mn-net.comDE / International:Tel.: +49 24 21 969-0Fax: +49 24 21 969-199 E-mail: [email protected]

CH:Tel.: +41 62 388 55 00 Fax: +41 62 388 55 05 E-mail: [email protected]

FR:Tel.: +33 388 68 22 68 Fax: +33 388 51 76 88 E-mail: [email protected]


www.mn-net.com





A031003/0180.25

Documents

RNA and DNA purification...2 Lyse cells 350 μL RA1, RAP, or RP1 3.5 μL reducing agent Mix 100 μL RA1 2 μL TCEP Mix 5 μL Carrier RNA 3 Filtrate ysatel 11,000 x g 1 min 11,000 x